The present invention relates to antigens associated with human breast cancer tumors. The present invention also relates to monoclonal antibodies which are specific to various determinants present on such breast tumor-associated antigens. The present invention further relates to a method of detecting the presence of breast cancer.
The early detection of cancer is both highly desireable and very difficult. The difficulty results primarily from the fact that there is no general marker for cancer which is 100% sensitive (no false negatives) and 100% specific (no false positives). Those markers which are known are deficient in both sensitivity and specificity. Moreover, such markers are not applicable to all types of cancer. For example, the best known marker, carcinoembryonic antigen (CEA), does not appear to be associated with breast cancer.
Breast cancer is the leading cancer in women and is among the three most common cancers generally. 1f breast cancer is detected sufficiently early, the prognosis for recovery is good. Thus, there is a continuing effort to discover breast cancer markers which will aid the physician in the early diagnosis of breast cancer.
One such effort is represented by U.S. Patent No. 4,383,985 to Bartorelli et al. which describes a series of antigens which are associated with breast cancer tumors. Such antigens were isolated from human primary breast carcinomas by known methods, usually by a combination of solvent extraction, ion-exchange and/or absorption chromatography, and gel filtration. When sufficiently pure, such antigens are not cross-reactive with anti-CEA antiserum and are stated to be extractable from human primary breast carcinomas with a glycoprotein solvent. Such antigens apparently are not associated with normal breast tissue.
In addition, a new marker for human cancer cells has been reported by Ashall et al. Lancet, 1982, ii: 1-6 and McGee et al., Lancet, 1982, ii: 7-10. Briefly, an antigen has been detected in the cell membranes of a wide variety of malignant human cell lines; the antigen was not detected in diploid human cell strains. The antigen was found in very low concentrations, if at all, in homogenates of normal adult or fetal tissues. It could be immunoprecipitated by a specified monoclonal antibody from extracts of malignant cells but not from extracts of nonmalignant cells. After reduction, the immunoprecipitated antigen separated in sodium dodecyl sulfate acrylamide into two bands having approximate molecular weights of 390,000 and 350,00 daltons, respectively. Both components appeared to be glycoproteins having a high carbohydrate content.
Moreover, a substantial body of work has been carried out by Ceriani and Taylor-Papadimitriou and their co-workers on human mammary epithelial antigens. While a complete discussion of such work is beyond the scope of this section, a synopsis of selected publications will serve as a substantive summary thereof.
Ceriani et al., Proc. Natl. Acad. Sci., 74, 582 (1977), describes surface differentiation antigens of human mammary epithelial cells carried on the human milk fat globule. Rabbit anti-human mammary epithelial cell antiserum was raised against the defatted human milk fat globule. By means of electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate, affinity chromatography employing the antiserum conjugated to Sepharose.TM. 4B, immunoflourescence staining, and indirect immunofluorescence staining, the antigenic components present on the human milk fat globule were characterized and/or separated.
The defatted human milk fat globule is composed of at least four main proteinaceous components, two of which appear to be glycoproteins. At least three of the four components are antigenic.
The antibodies raised against the defatted human milk fat globule appear to be organ-specific. The antigenic components appear to be lacking in cells other than breast epithelium since the antiserum does not bind to epithelial-like cells from kidney, lung, and colon.
The antigens detected by the antiserum are located on the breast epithelial cell surfaces and are the same as those located on the human milk fat globule membranes. Because the human milk fat globule is derived from the apical surface of the breast cell, the antigens may be restricted to this specialized surface.
The antigens continue to be expressed in breast carcinoma cell lines -and in metasteses of breast carcinomas. The antigenic expression, however, appears to be different for each breast tumor cell line.
Monoclonal antibodies to the above-described epithelium-specific . components of delipidated (defatted) human milk fat globule membrane are reported in Taylor-Papadimitriou et al., Int. J. Cancer, 28, 17 (1981). Cells from the spleens of mice sensitized to delipidated human milk fat globule were fused with cells from the myeloma line P3/NS1/1-Ag4-1. Three hybridomas were isolated which produced antibodies reactive with components of the delipidated human milk fat globule. Such reactivity, however, differed significantly among the three monoclonal antibodies produced by the hybridomas. The least reactive antibody appeared to bind only slightly. The reactivity of the most reactive antibody was approximately five times that of the least reactive antibody and a little less than twice that of the third antibody.
Two of the three monoclonal antibodies reacted with epithelial cells cultured from human milk and with seven of the eight breast cancer cell lines tested. The third monoclonal antibody was not reactive toward epithelial cells cultured from human milk and was reactive with only two of the eight breast cancer cell lines. None of the monoclonal antibodies reacted with any of the four fibroblast lines and strains tested, one of which was foreskin fibroblasts. The antigens reactive with the monoclonal antibodies appear to be either absent or present only in very low amounts on the eleven lymphoblastoid lines tested.
The seven epithelial cell lines tested were derived from human tumors, with the exception of two which were SV-40-transformed human keratinocytes and mouse mammary cells. The reactions of these cell lines with the three monoclonal antibodies were predominatly negative with a few exceptions. All three antibodies bound consistently but not strongly to a pharyngeal carcinoma line. One antibody bound to a colon carcinoma line and the other two antibodies showed binding to derivatives of HeLa, but not in every assay.
Two of the three monoclonal antibodies described above were assayed histologically, with the results being reported in Arklie et al., Int. J. Cancer, 28, 23 (1981). The histological assays consisted of an indirect immunoperoxidase staining technique against formalin-fixed, paraffin-embedded normal and tumor tissue sections and frozen sections fixed with 5% acetic acid in methanol. Neither antibody reacted with most of the epithelial cells in the resting breast. Those areas of the resting breast which did not stain always had intraluminal material which also stained. Both antibodies showed a strong positive reaction with epithelial cells and their secretion in the lactating breast. With respect to benign lesions, papillomas consistently showed strong postitive staining, whereas less than 10% of the epithelial element in fibroadenomas were stained positively.
One of the two monoclonal antibodies showed positive reactions with each of 20 primary breast carcinomas tested, and with metastatic lesions in lymph nodes from six of these. The other antibody also reacted with most of the primary carcinomas but not with those of the mucoid type or with metastatic lesions in lymph nodes. The only nonbreast tumors showing positive reactions were adenocarcinomas of the lung, ovary, and uterus. Other carcinomas, specifically those of the intestinal tract, cervix, nasopharynx, and liver, showed negative reactions.
One of both of the antibodies gave positive staining with normal tissue from the liver, pancreas, sebaceous gland, minor salivary gland, kidney, lung, sweat gland, epididymus, and uterus. Tissues showing negative staining with both antibodies included the stomach, small intestine, large intestine, appendix, thymus, thyroid, testis, fallopian tube, bladder, gall bladder, and skin.
An analysis of expression of human mammary epithelial antigens in normal and malignant breast cells at the single cell level by flow cytofluorimetry is reported in Peterson et al., Expl. Cell. Biol., 49, 1 (1981). Such analysis involved simultaneously labeling the cell surface with anti-human mammary epithelial cell membrane antiserum by indirect immunofluorescence and the cell DNA with propidium iodide. Upon eliminating the contribution of nonstaining breast cells to the distribution curves of fluorescent intensity, it was found that the relative binding of antiserum to breast epithelial cells from a normal breast and a fibrocystic disease of the breast was equal to or greater than that of the two breast cancer cell lines. When expressed per unit DNA, such relative binding was significantly higher than that of the two breast cancer cell lines.
A solid-phase radioimmunoassay to determine the presence of the human mammary epithelial antigens in sera is reported in Ceriani et al., Proc. Natl. Acad. Sci., 79, 5420 (1982). Using radiolabelled anti-human mammary epithelial cell membrane antiserum and total dilipidated human milk fat globule membrane as the antigen for construction of a standard curve, high levels of human mammary epithelial antigens were found in the sera of patients with disseminated breast cancer. Such levels were statistically significantly higher than the background levels (&lt;30 ng/ml) found in the sera of normal women and men and in female patients with benign breast disease, primary breast cancers, disseminated cancer of the lung, nervous tissue, and colon, and melanomas. By means of a three-step immunodetection method, three groups of antigens having molecular masses of 150,000, 70,000 and 46,000 daltons, respectively, were isolated from the sera of those patients showing high levels of human mammary epithelial antigens by the radioimmunoassay. Such antigens could not be isolated from the sera of patients having nonbreast tumors and normal sera, although a small amount of primarily nonspecifically bound human serum albumin was obtained. Similar immunodetection results were obtained when the Polyclonal antiserum was replaced with a monoclonal antibody directed to the 46,000-dalton human mammary epithelial antigen.
The above-noted monoclonal antibody and two others were described in Ceriani et al., Somatic Cell Genetics, 9, 415 (1983). Briefly, hybridomas that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells were prepared by fusion of mouse myeloma cells with spleen cells from mice or rats immunized with delipidated human milk fat globules. Three different monoclonal antibodies were produced and found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The highest molecular weight antigen appears to be a mucin-like glycoprotein having a high sugar content. By means of a radioimmunobinding assay, it was shown that all three monoclonal antibodies bound to human milk fat globule membranes and to four different breast cancer cell lines of epithelial origin. Such antibodies did not bind, however, to cells of 11 different nonbreast cancer lines or to normal breast fibroblasts. Levels of the highest molecular weight antigen were measured in three of the four breast cancer cell lines and were found to vary over a ten-fold range.
Two monoclonal antibodies to the human milk fat globule are discussed in Burchell et al., J. Immunol., 131, 508 (1983). Such two monoclonal antibodies are directed to antigenic determinants which appear to be tumor associated. The antigens are expressed on the lactating breast, but weakly, if at all, on the resting breast. Both antibodies recognize determinants found in human milk fat globule components having molecular weights greater than 400,000. However, the first antibody binds to delipidated human milk fat globule at a much lower concentration than the second, the difference apparently being between 10- and 100-fold. Similar differences in binding are seen with respect to human mammary epithelial cells and a breast cancer cell line, except that the relative levels of binding with the former cells, which are approximately equivalent to the binding levels with the human milk fat globule, are reversed with the latter cells. The first antibody reacted with high molecular weight components in human mammary epithelial cells which are similar to those in the human milk fat globule preparation. High affinity sites for the second were expressed by human mammary epithelial cells and a breast cancer cell line on several components of lower molecular weight. All other breast carcinoma lines examined and metastatic cells from two breast cancer patients expressed high affinity binding sites for the second antibody on components of varying size, i.e., from 80,000 to greater than 400,000. Only two of five cell lines and cancer cells from one of the two patients expressed high affinity sites for the first antibody, which sites were found on high molecular weight glycoprotiens, i.e., 300,000 to 400,000.
Three human mammary epithelial antigens, prepared from a membrane fraction of delipidated human milk fat globules, are the basis of a method for diagnosing the Presence of cancer in a mammalian host. See Published European Patent Application No. 0,080,259. The antigens involved include those described by Ceriani et al. having molecular weights of 48,000, 75,000, and 150,000, respectively. Simply stated, the method involves assaying a patient's plasma sample and looking for levels of one or more tumor-associated antigens which are elevated when compared to the levels present in normal individuals. When breast cancer is involved, such antigens are illustrated by the above-noted antigens described by Ceriani et al.
Finally, antigens and antibodies useful in the diagnosis and treatment of cancer are described in published U.K. Patent Application GB 2,121,417 A. The antigen was derived from malignant cells, specifically from cultured cells derived from a human laryngeal carcinoma. The antigen-has a molecular mass within the range of 340,000 to 400,000, the ability to bind to the lectin wheat germ agglutinin, resistance to boiling, and resistance to destruction and to extraction from malignant cells using certain specified solvents. Based on studies of the binding of monoclonal antibody to the antigen, the antigen appears to be present on the vast majority of malignant human tumor cells but not on the cells of benign tumors or on normal tissue cells.
Although progress is being made in the search for highly sensitive and specific cancer markers, including breast cancer markers, there still is a need for such a marker to aid in the diagnosis of breast cancer. It is to such need that the novel antigen of the present invention is directed.